New Benzil and Isoflavone Derivatives with Cytotoxic and NO Production Inhibitory Activities from Placolobium vietnamense

The phytochemical investigation of Placolobium vietnamense stems led to the isolation of a new isoflavone derivative (1) and three new benzil derivatives (2–4), together with four known pyranoisoflavones (5–8). The structures of all isolated compounds were determined on the basis of extensive spectroscopic analyses, including NMR and HRMS spectral data, as well as comparison of their spectroscopic data with those reported in the literature. The cytotoxicity of all isolated compounds was assessed against the human liver hepatocellular carcinoma (Hep G2) cell line, and compound 1 displayed the most significant cytotoxicity with an IC50 value of 8.0 μM. Furthermore, all isolated compounds were also tested for their inhibitory activity against NO production in RAW 264.7 macrophages. Of these, compound 1 exhibited the strongest inhibitory efficacy against the LPS-induced NO production with the IC50 value of 13.7 μM.


Introduction
Placolobium is a genus of plants in the family Fabaceae, which contains three accepted species. These are distributed throughout the world's tropical regions, some extending into temperate zones, especially in East Asia [1]. Placolobium vietnamense N.D.Khoi & Yakovlev is an indigenous plant species, known in Vietnam as 'Rang Rang'. It is a perennial tree with a straight, cylindrical trunk, and brown bark. The fruit is a small pod with a single seed. This plant is used as a folk remedy for snakebites, debility, and to increase strength after childbirth [1]. There has only been one investigation into the chemical constituents of P. vietnamense [1]. Previously, our group reported the isolation and structure elucidation of six isoflavonoids, including afrormosin, cladrastin, 8-O-methylretusin, millesianin C, barbigerone, and durallone from the EtOAc stem extract of this plant, together with their cytotoxicity. Encouraged by structurally diverse bioactive compounds from Placolobium species [2], the aim of this investigation is to revisit P. vietnamense in order to search for new bioactive compounds. We report herein the isolation and characterization of benzil and isoflavone derivatives from the stems of P. vietnamense. All isolated compounds were assessed for their cytotoxicity against human liver hepatocellular carcinoma (Hep G2) cell line, which is one of the most fatal cancers and has spread to the liver from other organs. Additionally, the inhibitory activity toward NO production in RAW 264.7 macrophages of all isolated compounds was also evaluated.

Structural Elucidation of the Isolated Compounds
Chromatographic separation of benzil and isoflavone derivatives from P. vietnamense stems allowed for the isolation of eight compounds, including a new pyranoisoflavone, placovinone A (1), and three new benzil derivatives, placovinones B-D (2-4), together with four known pyranoisoflavones (5-8) (Figure 1). The structures of all isolated compounds were elucidated based on NMR and HRMS spectral data and a comprehensive comparison of their spectroscopic and physical data with values from the published literature. The known isolated pyranoisoflavones were characterized as ichthynone (5) [3], durmillone (6) [4], calopogoniumisoflavone B (7) [5], and 4′,5′dimethoxy-6,6-dimethylpyranoisoflavone (8) [6]. Compound 1 was isolated as a colorless gum. The HRESIMS revealed a protonated molecular ion peak at m/z 367.1549 [M + H] + (calcd for C22H23O5 367.1545) corresponding to the formula C22H22O5. The 1 H NMR signal at δH 8.42 (1H, s, H-2) and 13 C NMR signal at δC 152.7 (C-2) were characteristic of the isoflavone skeleton [7]. The existence of AA′BB′ spin-system indicated para-substituted B-ring. The presence of a 2,2-dimethyldihydro pyran [8] and two methoxy substituents was identified from both 1 H and 13 C NMR spectra (Table 1). A singlet at δH 7.32 (1H) was assigned to the aromatic proton H-5 on the basis of the long-range coupling to C-4 (δC 174.4), C-7 (δC 148.3), and C-8a (δC 109.9), observed in the HMBC spectrum ( Figure 2). The methoxy group at δH 3.84 (3H, s) was assigned as 6-OCH3 according to the HMBC correlation between 6-OCH3 and C-6 (δC 147.1). The longrange correlations observed in the HMBC spectra of H-1′′ at δH 2.87 (2H, t, J = 6.5 Hz) to C-7 and C-8a were key correlations that revealed the position of 2,2-dimethylpyran moiety was fused to C-7 and C-8. Its 1D and 2D NMR spectral data were similar to those of 6-methoxycalopogonlum isoflavone A [9], except for the replacement of a double bond at C-1′′ and C-2′′ of the 2,2-dimethylpyran substituent in 6-methoxycalopogonlum isoflavone A by a C-C single bond in 1. Based on the above spectral evidence, the structure of 1 was established and trivially named as placovinone A.   13 C NMR signal at δ C 152.7 (C-2) were characteristic of the isoflavone skeleton [7]. The existence of AA BB spinsystem indicated para-substituted B-ring. The presence of a 2,2-dimethyldihydropyrano [8] and two methoxy substituents was identified from the 1 H and 13 C NMR spectral data (Table 1). A singlet resonance at δ H 7.32 was assigned to the aromatic proton H-5 on the basis of the long-range coupling to C-4 (δ C 174.4), C-7 (δ C 148.3), and C-8a (δ C 109.9), observed in the HMBC spectrum ( Figure 2). The methoxy group δ H 3.84 (s) was assigned as 6-OCH 3 according to the HMBC correlation between 6-OCH 3 and C-6 (δ C 147.1). The longrange correlations observed in the HMBC spectrum of H-1" (δ H 2.87, t, J = 6.5 Hz) to C-7 and C-8a were key correlations that revealed the position of 2,2-dimethyldihydropyrano moiety was fused to C-7 and C-8, with the anticipated oxygenation at C-7 being supported by the HMBC correlation from H-5 to C-7. Its 1D and 2D NMR spectral data were similar to those of 6-methoxycalopogonlum isoflavone A [9], except for the replacement of a double bond at C-1" and C-2" of the 2,2-dimethylpyrano substituent in 6-methoxycalopogonlum isoflavone A by a C-C single bond in 1. Based on the above spectral evidence, the structure of 1 was established and trivially named as placovinone A.   Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C23H24O8 based on a protonated molecular ion peak at m/z 429.1566 (calcd for C23H25O8 429.1549). The signal of a hydroxyl group at δH 10.11 (1H, s, 2-OH) in the 1 H NMR spectrum, together with those of two carbonyl groups at δC 190.7 (C-7) and 191.4 (C-8) in the 13 C NMR spectrum, indicated that 2 was a derivative of 1,2-diphenyl-1,2-ethanedione [10]. The 1 H and 13 C NMR spectral data ( Table 2) further revealed the presence of a 2,2-dimethylpyran fragment and four methoxy substituents. In the 1 H NMR spectrum, two singlet protons at δH 6.76 and 7.41, were assigned to the two para-positioned aromatic protons H-3'and H-6' of the B-ring [11], indicating the B-ring of 2 with Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C 23 H 24 O 8 based on a protonated molecular ion peak at m/z 429.1566 (calcd for C 23 H 25 O 8 429.1549). The signal of a hydroxyl group at δ H 10.11 (s, 2-OH) in the 1 H NMR spectrum, together with those of two carbonyl groups at δ C 190.7 (C-7) and 191.4 (C-8) in the 13 C NMR spectrum, indicated that 2 was a derivative of 1,2-diphenyl-1,2ethanedione [10]. The 1 H and 13 C NMR spectral data ( Table 2) further revealed the presence of a 2,2-dimethylpyrano fragment and four methoxy substituents. In the 1 H NMR spectrum, two singlet protons at δ H 6.76 and 7.41, were assigned to the two para-positioned aromatic protons H-3' and H-6' of the B-ring [11], indicating the B-ring of 2 with 2 ,4 ,5 -trimethoxy substituent. This was also supported by the strong correlations in the HMBC spectrum ( Figure 2). The singlet of the aromatic proton at δ H 7.24 was identified as H-6 on the basis of the HMBC correlations from H-6 to C-1 (δ C 112.5), C-2 (δ C 149.7), C-4 (δ C 148.2), and C-7 (δ C 190.7). Consequently, the remaining methoxy group (δ H 3.84, s) was located at C-5, confirmed by the key HMBC correlation between 5-OCH 3 and C-5 (δ C 142.5). Hence the location of the 2,2-dimethylpyrano moiety was found to be at C-3 (δ C 108.9) and C-4, with the anticipated oxygenation at C-4 being confirmed by the HMBC correlation from H-6 to C-4. A careful comparison of the 1 H and 13 C NMR spectral data ( Table 2) of 2 with dielsianone [12] identified similar signals, distinguished by the presence of two methoxy groups at C-2 and C-5 . The existence of these two methoxy substituents was confirmed by the HMBC correlations from 2 -OCH 3 (δ H 3.33, s) and 5 -OCH 3 (δ H 3.89, s) to C-2 (δ C 156.8) and C-5 (δ C 155.6), respectively (Figure 2). From the aforementioned results, the structure of 2 was identified and named as placovinone B. . This was further confirmed by the 13 C NMR spectral data, which disclosed one methylene, two methyl, two olefinic, three aromatic methine, four methoxy, and ten quaternary carbons. The spectroscopic 1 H and 13 C NMR patterns of 3 (Table 2) were very similar to those of 2, with the only difference being that the keto carbonyl group at C-8 in 2 (δ C 191.4) was replaced by a methylene substituent in 3. This deduction was supported by the HMBC correlations from H-8 (δ H 4.20, s) to C-7 (δ C 202.9) and C-1 (δ C 114.2). Based on the above spectral evidence, compound 3 was identified and named placovinone C.
Compound 4 was obtained as a white amorphous powder. The molecular formula C 21 H 22 O 5 was obtained from its HRESIMS, which showed a protonated molecular ion peak at m/z 355.1553 [M + H] + (calcd for C 21 H 23 O 5 355.1545). 13 C NMR and HSQC spectra of 4 indicated 21 signals, including one carbonyl, one methylene, two methyl, two methoxy, seven methine, and eight quaternary carbons. Two signals at δ H 7.21 (d, J = 8.7 Hz, H-2 , 6 ) and 6.88 (d, J = 8.7 Hz, H-3 , 5 ) appearing as an AA BB type confirmed the presence of a simple para-substituted B-ring, with a methoxy group (δ H 3.47, s) being positioned at C-4 (δ C 158.2). The careful comparison of the 1 H and 13 C NMR spectral data ( Table 2) of 4 was shown to be similar to those of 3, differing only in the absence of two methoxy groups at C-2 (δ C 130.7) and C-5 (δ C 114.1) on the B-ring of 4, which was supported by the COSY and HMBC correlations (Figure 2). On the basis of these spectral data, the structure of 4 was unambiguously established and named as placovinone D.

Cytotoxicity
The cytotoxicity of each isolated compound against Hep G2 cell line was assessed [13][14][15] and the IC 50 values are listed in Table 3.

Inhibition of Nitric Oxide Production
To determine the inhibitory effects of the isolated compounds on NO production (Table 3), LPS-stimulated RAW 264.7 cells were treated with various concentrations of tested compounds [16]. Additionally, the viability of RAW 264.7 cells using an MTT assay to avoid the cytotoxic effects of the isolated compounds was evaluated. Among eight isolated compounds, compounds 1 and 4 highly inhibited NO production in RAW 264.7 cells with the IC 50 values of 13.7 and 15.5 µM, respectively, whereas compounds 2, 3, and 8 moderately inhibited NO production with the IC 50 values of 31.0, 47.4, and 54.7 µM, respectively. Compounds 1 and 4 demonstrated cytotoxicity toward RAW 264.7 cells with the IC 50 values of 79.2 and 42.6 µM, respectively, while most of the other compounds showed no obvious cytotoxicity (IC 50 >100 µM). These results demonstrate that the presence of the para-substituted B-ring of 1 and 4 might be responsible for inhibiting NO production.

General Experimental Procedures
The NMR spectra were recorded on Bruker AvanceNEO 600 MHz and Bruker Avance III™ HD 500 MHz NMR spectrometers in DMSO-d 6 (Merck, Darmstadt, Germany). Optical rotations were measured on a A.KRÜSS Optronic P8000 polarimeter (KRÜSS, Hamburg, Germany). The IR data were obtained with a Jasco 6600 FT-IR spectrometer using an ATR technique (Jasco, Japan). The HRESIMS spectral data were generated with a X500 R QTOF model mass spectrometer (Sciex, Framingham, MA, USA) and Dionex Ultimate 3000 HPLC system hyphenated with a QExactive Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific, Waltham, MA, USA). Silica gel 70-230 mesh (Merck) and Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography.

Plant Material
The stems of P. vietnamense were collected in Dak Nong province, Vietnam, in February 2017. The plant material was identified by botanist Vo Van Chi (former lecturer at the University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam). A voucher specimen (No. SGU-A001) has been deposited in the Herbarium of the Laboratory of Chemistry-Biology-Environment, Sai Gon University, Ho Chi Minh City, Vietnam.

Cytotoxicity Assay
According to a previous procedure [17], the cytotoxic evaluation of 1-8 against the growth of human hepatocellular carcinoma (Hep G2) cell line was carried out. The positive control was ellipticine, a powerful anticancer medication with various modes of action. The cancer cells were grown in Dulbecco's Modified Essential Medium (DMEM) at 37 • C in a 5 % CO 2 environment with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, and 1% L-glutamine. The investigated compounds were added at concentrations ranging from 0.5 to 128 µg/mL by dissolving in DMSO (20 mg/mL), and the incubation was carried out once more for 72 h under the same conditions. Following the procedure, an MTT solution (10 µL, 5 mg/mL) was added to each well. The percentage of cell viability vs. sample concentration was plotted using SigmaPlot 10 (Systat Software Inc., San Jose, CA, USA) to calculate the IC 50 values.
3.5. Inhibition of Nitric Oxide Production Assay 3.5.1. Cell Culture RAW 264.7 cells were stocked in Dulbecco's Modified Essential and grown at the condition of 37 • C in DMEM supplemented with 10% heat-inactivated FBS, streptomycin sulfate (100 µg/mL), and penicillin (100 units/mL) in a humidified environment of 5% CO 2 . The RAW 264.7 cells were pre-incubated every two days.

Cell Viability Assay on RAW 264.7 Cells
The cell viability assay was used to determine the cytotoxic effect of the isolated compounds on RAW 264.7 cells. At a density of 1 × 10 5 cells per well, RAW 264.7 cells were seeded on a 96-well plate and allowed to adhere for 4 h. Then, the cells were treated with 0.5% DMSO, celastrol, and isolated compounds at the indicated concentrations. Celastrol was used as a positive control [16]. After incubating 24 h, the viable cells were measured with a colorimetric assay based on the mitochondria's ability in viable cells to reduce MTT [18]. The viability cells were treated with vehicle only and were defined as 100% viable. [OD 570 (treated cell culture) × 100]/OD 570 was the formula used to determine the percentage of macrophage surviving cells after treatment (vehicle control).

Measurement of Nitric Oxide (NO) Production
The RAW 264.7 cells were stimulated with or without 1 µg/mL of LPS (lipopolysaccharide), which was purchased from Sigma Chemical Co. (St. Louis, MO, USA), for 24 h with or without 0.5% DMSO, celastrol, and isolated compounds at the indicated concentrations. The culture supernatant (100 µL) was then reacted with 100 µL of Griess reagent [16]. After the Griess assay, the remaining cells were used to screen for their viability using colorimetric assay-MTT (Sigma Chemical Co., St. Louis, MO, USA).

Conclusions
In conclusion, we have conducted the successful isolation of eight compounds, including a new isoflavone derivative (1) and three new benzil derivatives (2)(3)(4), together with four known pyranoisoflavones (5-8) from P. vietnamense stems. To the best of our knowledge, compounds 1-8 were isolated for the first time from the genus Placolobium. The biological evaluations showed that 1 exhibited the most significant cytotoxicity toward Hep G2 cell line and the strongest inhibitory activity against the LPS-induced NO production. According to these investigation results, the structure of 1 is a promising candidate and could be used as a template for discovering potential anticancer and anti-inflammatory agents.